Gut-Testis Axis: Microbiota Prime Metabolome To Increase Sperm Quality in Young Type 2 Diabetes

ABSTRACT Young type 2 diabetes (T2D) affects 15% of the population, with a noted increase in cases, and T2D-related male infertility has become a serious issue in recent years. The current study aimed to explore the improvements of alginate oligosaccharide (AOS)-modified gut microbiota on semen quality in T2D. The T2D was established in young mice of 5 weeks of age with a blood glucose level of 21.2 ± 2.2 mmol/L, while blood glucose was 8.7 ± 1.1 mM in control animals. We discovered that fecal microbiota transplantation (FMT) of AOS-improved microbiota (A10-FMT) significantly decreased blood glucose, while FMT of gut microbiota from control animals (Con-FMT) did not. Sperm concentration and motility were decreased in T2D to 10% to 20% of those in the control group, while A10-FMT brought about a recovery of around 5- to 10-fold. A10-FMT significantly increased small intestinal Allobaculum, while it elevated small intestinal and cecal Lactobacillus in some extent, blood butyric acid and derivatives and eicosapentaenoic acid (EPA), and testicular docosahexaenoic acid (DHA), EPA, and testosterone and its derivatives. Furthermore, A10-FMT improved liver functions and systemic antioxidant environments. Most importantly, A10-FMT promoted spermatogenesis through the improvement in the expression of proteins important for spermatogenesis to increase sperm concentration and motility. The underlying mechanisms may be that A10-FMT increased gut-beneficial microbes Lactobacillus and Allobaculum to elevate blood and/or testicular butyric acid, DHA, EPA, and testosterone to promote spermatogenesis and thus to ameliorate sperm concentration and motility. AOS-improved gut microbes could emerge as attractive candidates to treat T2D-diminished semen quality. IMPORTANCE A10-FMT benefits gut microbiota, liver function, and systemic environment via improvement in blood metabolome, consequently to favor the testicular microenvironment to improve spermatogenesis process and to boost T2D-diminished semen quality. We established that AOS-improved gut microbiota may be used to boost T2D-decreased semen quality and metabolic disease-related male subfertility.

I thank the authors for performing this research on the influence of alginate oligosaccharide (AOS) supplemented gut microbiota on sperm quality in type 2 diabetes. I appreciate the effort put into the various experiments to get the results presented here. It seems a bit of care needs to be taken in the interpretation of some of the results of this research.
A few issues need to be addressed: Lines 55-56: A10-FMT increased the gut 'Lactobacillus and Allobaculum'. However, Figure 1d (Allobaculum) shows no significant difference between the A10-FMT and Con-FMT. Similarly, Figure 1d (Lactobacillus) shows no significant differences across all groups. So, I think this cannot be attributed to the A10-FMT. Your results show that both genera were present in the groups tested though to different levels. Figures 1a, b, c, and d are missing the 'Con' group. It would be nice to see what groups of bacteria were in this group on a normal diet and the relative amounts of the different genera along with the other groups. Though from Figure S1, looks like similar amounts of Allobaculum and Lactobacillus were present in the Con and HS groups Lines 293-294: Here you are contradicting the results presented in Figure 1d on Allobaculum as there was no significant difference between the A10-FMT and Con-FMT groups. So, you will need to reword or get rid of the statement.
In general, from the results in Figure 1d, it will be erroneous to attribute the change in microbiota to Allobaculum and Lactobacillus alone as these genera were not exclusively present in this group only. I would suggest you look closely at your microbiota data to check if there are any differences attributable to specific genera for A10-FMT where it is significantly different from HS and Con-FMT. For example, members of the Ruminococcus genus are also short-chain fatty acid (SCFA) producers.
Lines 295-296: Looks like there is another contradiction here as Figure 3i shows similar levels of blood butyric acid in A10-FMT and Con-FMT. Was there a significant difference between the butyric acid from the A10-FMT vs Con-FMT groups? I assume not as there is no indication on the figure since you did indicate between the Con and HS groups. While I am not doubting the correlation between SCFA production and an increase in sperm concentration and motility, this must be rightly attributed to ensure reproducibility.
Figures 3d, h, I, and l: Important to compare A10-FMT vs Con-FMT to determine any significant differences. Please indicate using asterisks or ns Line 67: Change 'benefited' to 'improved' in AOS-benefited gut microbiota (A10-FMT) Line 105: Think 'as' is missing after 'such' Line 111: Write R in Rhus coriaria in full as it appears once Line 122. Please add a reference at the end of the sentence 'busulfan treated subjects' Lines 140-141: Here you need to perform a comparison between the HS and the Con-FMT in Figure 1b before you can say it was not significantly different as you did for the glycogen in Figure 1c. This is because you indicated in Figure 1b, that there is no significant difference between A10-FMT vs Con-FMT. Lines 149-150: Same issue as discussed previously with Allobaculum. Since the aim of this study is to show that AOS supplemented microbiota contributed to these findings, I wonder if it is not necessary to compare with the Con-FMT here. I mean, is it possible that the increase in Allobaculum is because of the FMT process and not because of the AOS since no significant differences in Allobaculum were observed in both groups (A10-FMT vs Con-FMT)?
Lines 229-233: Looks like no comparison was performed between the HS and Con-FMT here. For Figures 5a to 5p, I think it is important to see if any significant difference exists between these groups as shown in 5c and 5i. The reason behind this is because for some of these e.g., Figures 5e, m, o, and p, there were no significant differences between the A10-FMT and Con-FMT groups. Line 718: Should be 'mmol/L' not mmlo/L Lines 727-739, 760-786. After writing the main legend, rather than repeating what the x and y axes are for each, you could write; For Figures, 3g, h, I, j, k, l, m, and n, the y-axis represents the relative amount and the x-axis represents the treatment, *p < 0.05 for all. This would save a lot of space and repetition as this is already shown in the Figures. The same applies to Figures 5c,d,e,f,g,h,I,l,m,n,o,and p. Similarly,legends for Figures 6a,b, (1) General considerations It would be useful to know what statistical test was used for each of the figures, that is, for example, which of the data sets was the T-test used for? Since most of the figures are compared across three or four groups and appropriate tests with corrections for multiple comparisons are ideal. Though it is stated in the methods section, it is not clear which tests were used for which analysis. Consider adding, 'one-way ANOVA was used with LSD for multiple comparisons'. I wonder what the actual p values were as I suppose this was obtained during the analysis as the differences between some assays are different from others e.g Figure 5A on DHA, the statistical difference between HS and A10-FMT was it less than p < 0.01 or p < 0.05? The number of the figure ought to be indicated on each figure to make it easier to navigate since the figure legends are elsewhere.
Here is a paper on Allobaculum and its potential role in obesity: Frontiers | Allobaculum Involves in the Modulation of Intestinal ANGPTLT4 Expression in Mice Treated by High-Fat Diet | Nutrition (frontiersin.org) You may also want to look at this: Identification of Allobaculum mucolyticum as a novel human intestinal mucin degrader -PMC (nih.gov) Reviewer #3 (Comments for the Author): The work "Gut-testis axis: microbiota prime metabolome to increase sperm quality in young type 2 diabetes", conducted by Dr. Xiaowei Yan and colleagues, enriches the field of male infertility and microbiota. The authors were able to respond elegantly and robustly to the issues that motivated the study. Using a murine model of type 1 diabetes, the authors were able to demonstrate that the transplantation of alginate oligosaccharide (AOS)-modified gut microbiota was able to significantly decreased blood glucose and recover 5 to 10 times the concentration and motility of the subjects' sperm.

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Review manuscript "Gut-testis axis: microbiota prime metabolome to increase sperm quality in young type 2 diabetes"

Summary
The authors want to test the effect of AOS-gut modified microbiota by means of fecal microbiota transplantation on a type 2 diabetes phenotype in mice (A10-FMT). They induced a T2D phenotype by a high fat diet and injection of streptozotocin. They then treated mice with A10-FMT. They compared these to control animals, animals with an untreated T2D phenotype and animals with T2D but treated with control FMT (Con-FMT).
They determined a T2D phenotype by measuring blood glucose.
They assessed the effects of the different types of FMT by examining microbiota in different parts of the intestine, sperm quality, liver function, testicular metabolome and microenvironment, and spermatogenesis.
They claim that: 1. A10-FMT significantly decreased blood glucose 2. T2D shows sperm concentration and motility decrease 3. A10-FMT improved liver functions and systemic anti-oxidant environment 4. A10-FMT promoted spermatogenesis through the improvement in the expression of proteins important for spermatogenesis to increase sperm concentration and motility The suggest that the potential mechanism is: "A10-FMT increased gut beneficial microbes Lactobacillus and Allobaculum to elevate blood and/or testicular butyric acid, DHA, EPA, and testosterone to promote spermatogenesis and thus to ameliorate sperm concentration and motility".
Throughout the results sections they make summarizing claims of what their data supports, which are partly correct and partly not. In the following sections I make suggestions on how this can be improved so that the true changes observed are highlighted.

Major items
Test effectivity of T2D treatment with FMT on male fertility by performing fertility tests such as examining how many pregnancies come to term and offspring health status. This is particularly important as the authors state that T2D is linked to various symptoms of male infertility, not confined to sperm count and motility.
SupFig 4a-b are unsupported by the data. There is no change in either AST ALT by introducing the T2D phenotype so any changes with the treatment and likely not related. Provide stats between the control groups and A10-FMT group. Where ALT is (significantly?) decreased in A10-FMT samples compared to Con and discuss that this could be a sign for vitB deficiency or chronic kidney disease.
Line 200: AST was "increased" in HS; it is not significant and could be just a trend. Provide stats. By the same means one could state that HS has decreased ALT compared to control.
FigS4f-state is H&E and must be corrected in text line 211: "The protein level of NR1H3 was increased in HS while it was decreased in A10-FMT but not in Con-FMT (Fig. 4a, f), which is consistent with the histopathology of the liver samples in different groups (Fig. S4f) Fig3b stats between control land A10-FMT being non-significant would strengthen the statements made.
Fig 3D stats between control and HS is needed here again. The reason is that one can establish the phenotype of T2D and therefore the effects of any treatment on T2D ONLY when there is significance between control and HS (untreated T2D model).
Line 191 "Similarly, blood retinoic acid was decreased in HS, however, it was increased in A10-FMT (Fig. 3m)." needs to be rephrased to reflect the lack of significance. One can state trends and then in  A potential treatment cannot just reflect a difference to the pathology but has to reflect similarity to the WT because there can also be adverse effects and side effects to each treatment which need to be made known for consideration.
Section "A10-FMT improved the testicular metabolome and the testicular microenvironment" needs to be re-written to reflect the effects between wt and HS groups as well. For example, the paper states that retinoids are important for spermatogenesis and that those are elevated in A10-FMT, but they are much higher than the wildtype and more importantly the HS group does not seem to have a phenotype of reduced retinoids which signals that the specific measurement is not relevant to T2D.
The statement "HS treatment significantly decreased these hormones while A10-FMT led to a recovery (Fig. 6a-c)." is not true for Fig6c .   Fig 6f-g, again, it is not clear to me how these data were collected, and the conclusions made. Which group was used to make this analysis? Was it control to the HS groups? This information needs to be clear in the text and in the figure legend.
Figs 1d and 7d are conflicting with respect to the con-FMT data. Please discuss.

Fig 1f
No significance between control and HS for TP1 and PIWIL1. Please rephrase in the text (lines 257-8). I would also recommend trying tubulin as a control because it is seems that actin was overloaded and may not have served as an adequate loading control.

Minor items
Line 46: edit to something like "Young type diabetes 2 affects x% of population with a noted increase in cases in recent years". Line 47: "aimed at exploring" Line 47: "beneficial improvements" is redundant. Remove "beneficial" or write "beneficial effects" Replace "youth mice" with "young mice". "of 5 weeks of age". Would be good to explain why the measurement of blood glucose level is enough to determine a model of T2D has been generated. Why HbA1C% test was not done, what is the speed of induced type1D development in mice differs to that of T2D. They could for example show that in their model T2D mice do express insulin, a key difference between T2D and T1D.
Line 367: state where streptozotocin was injected (ie. IP, IM, IV?). Line 134 & 136: add the use of streptozotocin for a complete description of the study group of the FMT treated groups Include Con-FMT schematic in Figure 1.

FigS1a -why is BW reduced in High fat diet mouse groups?
Fig S1b -what do point represent? 30 mice are stated per group but only 4-5 points appear on the graphs. Table S1 -protein size for TP1 is 6.4kDa Line 226 "Since A10-FMT improved liver functions and the blood metabolome, next we set out to explore the beneficial advantages of A10-FMT on the testicular metabolome". The sentence is leading by stating beneficial effects on the testicular metabolome prior to showing the supportive data. I would remove the term beneficial at this point and welcome the reader to decide as the results are presented.
Line 256, stats not significant for SYCP3 so the statement is unsupported.
Based on Fig 7 con-FMT treatment seems to reverse the sperm quality problems and would resolve the specific part of the phenotype.
In the material and methods, perhaps include a statement as to why male mice were used and not females (hormone fluctuations etc).
The material and methods include a section on acrosome integrity which was not talked about in the paper or figures.
Line 512, how many sections were analysed?
It might interesting to discuss how long the effects of FMT treatment would last.

Response to reviewer's comments
First, we would like to thank the editors and reviewers very much for the comments.

Reviewer(s)' Comments to Author:
Reviewer #1 (Comments for the Author): Very happy to see such a collaborative effort which is why the five shared first authors is explained. The text would benefit from being edited. I started making edits but it soon became obvious that it would take too long within the review time window.
I can however, do it if you wish so. It really is not much but the text would benefit at places. I have made some suggestions which might prove useful to you in the review document.

Attachment:
Review manuscript "Gut-testis axis: microbiota prime metabolome to increase sperm quality in young type 2 diabetes"

Summary
The authors want to test the effect of AOS-gut modified microbiota by means of fecal microbiota transplantation on a type 2 diabetes phenotype in mice (A10-FMT). They induced a T2D phenotype by a high fat diet and injection of streptozotocin. They then treated mice with A10-FMT. They compared these to control animals, animals with an untreated T2D phenotype and animals with T2D but treated with control FMT (Con-FMT).
They determined a T2D phenotype by measuring blood glucose.
They assessed the effects of the different types of FMT by examining microbiota in different parts of the intestine, sperm quality, liver function, testicular metabolome and microenvironment, and spermatogenesis.
They claim that: The suggest that the potential mechanism is: "A10-FMT increased gut beneficial microbes Lactobacillus and Allobaculum to elevate blood and/or testicular butyric acid, DHA, EPA, and testosterone to promote spermatogenesis and thus to ameliorate sperm concentration and motility".
Throughout the results sections they make summarizing claims of what their data supports, which are partly correct and partly not. In the following sections I make suggestions on how this can be improved so that the true changes observed are highlighted.

Reviewer #2 (Comments for the Author):
I thank the authors for performing this research on the influence of alginate oligosaccharide (AOS) supplemented gut microbiota on sperm quality in type 2 diabetes. I appreciate the effort put into the various experiments to get the results presented here. It seems a bit of care needs to be taken in the interpretation of some of the results of this research.
A few issues need to be addressed: Comment #1: Lines 55-56: A10-FMT increased the gut 'Lactobacillus and Allobaculum'. However, Figure 1d (Allobaculum) shows no significant difference between the A10-FMT and Con-FMT. Similarly, Figure 1d (Lactobacillus) shows no significant differences across all groups. So, I think this cannot be attributed to the A10-FMT. Your results show that both genera were present in the groups tested though to different levels.
Response: Thank the reviewer very much for the very nice comment. In small intestine, compared to HS group A10-FMT increased Allobaculum significantly, however Con-FMT increased Allobaculum in some extent (not significant). And there was no significant difference between A10-FMT and Con-FMT.
In small intestine, A10-FMT and Con-FMT similarly increased Lactobacillus in some extent (not significant) compared to HS group.
In cecum, compared to HS group, A10-FMT and Con-FMT increased Lactobacillus in some extent (not significant), while it was slightly higher in A10-FMT than Con-FMT (not significant). We revised the abstract as the reviewer suggested. Thanks. Figures 1a, b, c, and d are missing the 'Con' group. It would be nice to see what groups of bacteria were in this group on a normal diet and the relative amounts of the different genera along with the other groups. Though from Figure S1, looks like similar amounts of Allobaculum and Lactobacillus were present in the Con

and HS groups
Response: Thank the reviewer very much for the very nice comment. The comparation of bacteria for Con group and HS group (small intestine, cecum, colon) are present in Fig. S1e, f, i, j, m, n. In order to make the data clear for A10-FMT and Con-FMT compared to HS group, the data for Con group were not added in Fig 2a, b, c and d (the reviewer mentioned as Fig. 1a, b, c, and d). Yes, the amounts of Allobaculum and Lactobacillus were similar in the Con and HS groups.
Helicobacter was increased in cecum and colon, which was decreased by A10-FMT and/or Con-FMT. Thanks.

Comment # 3:
Lines 293-294: Here you are contradicting the results presented in Figure 1d on Allobaculum as there was no significant difference between the A10-FMT and Con-FMT groups. So, you will need to reword or get rid of the statement.
Response: Thank the reviewer very much for the very nice comment. We have revised it as the reviewer suggested. Thanks.

Comment # 4:
In general, from the results in Figure 1d, it will be erroneous to attribute the change in microbiota to Allobaculum and Lactobacillus alone as these genera were not exclusively present in this group only. I would suggest you look closely at your microbiota data to check if there are any differences attributable to specific genera for A10-FMT where it is significantly different from HS and Con-FMT. For example, members of the Ruminococcus genus are also short-chain fatty acid (SCFA) producers.
Response: Thank the reviewer very much for the very nice comment. As we responded in Comment #1, there are a few microbes were changed by A10-FMT significantly while altered by Con-FMT not significantly. The effects of A10-FMT may be multiple effects from a few different microbes. We revised the manuscript to make it more clearly. Thanks.

Comment # 5:
Lines 295-296: Looks like there is another contradiction here as Figure 3i shows similar levels of blood butyric acid in A10-FMT and Con-FMT. Was there a significant difference between the butyric acid from the A10-FMT vs Con-FMT groups? I assume not as there is no indication on the figure since you did indicate between the Con and HS groups. While I am not doubting the correlation between SCFA production and an increase in sperm concentration and motility, this must be rightly attributed to ensure reproducibility.
Response: Thank the reviewer very much for the very nice comment. Yes, although the level of butyric acid was higher in A10-FMT, the difference was not significant between A10-FMT and Con-FMT. We revised the text as the reviewer suggested. Thanks.

Response to reviewer's comments
First, we would like to thank the editors and reviewers very much for the comments.

Reviewer(s)' Comments to Author:
Reviewer #1 (Comments for the Author): Thank you for revising the manuscript.
There are a few remarks on changes made and some minor edits. There is also a comment that was not addressed. You will find all suggestions below: Please refer to this in the discussion stating that to claim improve sperm quality one must indeed document a significant increase in pregnancies that come to term.
Response: Thank the reviewer very much for the very nice comment. We added the statement in the discussion section as the reviewer suggested "In current investigation, sperm motility and concentration were increased by A10-FMT compared to T2D (HS group), however, the pregnancy rate was not determined". Thanks.  Fig. 1c and revised the text as the reviewer suggested. Thanks.
Please remove Fig 3D as the difference between HS and Con is not significant, so any data is not representative of the model the authors present.
Response: Thank the reviewer very much for the very nice comment. Fig 3d was deleted as the reviewer suggested. Thanks.
Comment (R2) #5: Comment #12: Line 191 "Similarly, blood retinoic acid was decreased in HS, however, it was increased in A10-FMT (Fig. 3m)." needs to be rephrased to reflect the lack of significance. One can state trends and then in Fig4 where the protein synthesis of retinoic acid is discussed, remind the reader of Fig 3m. Response: Thank the reviewer very much for the very nice comment. We revised the text as the reviewer suggested. Thanks.
Line 193 Please add the following: 3 "It should be noted that in some cases the data were not significant between Con and HS (Fig 3k-m), which suggests that the changes observed may not be representative for T2D." Response: Thank the reviewer very much for the very nice comment. It was added as the reviewer suggested. Thanks. Thank the reviewer very much for the very nice comment. We added stats for Fig. 4j and revised the text as the reviewer suggested. Yes, the data were significant between Con and HS for GPX1. We revised the text. We have tried to make the graph clear for Fig. 4 i, however, if all the data shown in one graph, it is hard to make it very simple.

Comment
Thank the review for the understanding. Thanks.
Line 223 Please edit the text to: "Con and HS for some of the compounds (Fig. 5a, c, f, I, k, n, o, p)." AND "... however, the data were not significant between Con and HS which suggests that the changes observed may not be representative for T2D." Response: Thank the reviewer very much for the very nice comment. It was added as the reviewer suggested. Thanks. potential treatment cannot just reflect a difference to the pathology but has to reflect similarity to the WT because there can also be adverse effects and side effects to each treatment which need to be made known for consideration. Response: Thank the